1. Field of the Invention
The present invention is related to assays for identifying potential carcinogens. More specifically, the present invention provides a transgenic animal model in which to test potential carcinogens.
2. Description of the Related Art
Assays for identifying potential carcinogens are well known to those skilled in the art; see, e.g., U.S. Pat. No. 4,997,757, the entire disclosure of which is hereby incorporated by reference into this specification.
The assay described in U.S. Pat. No. 4,997,757 utilizes a viable strain of the unicellular yeast Saccharomyces cerevisiae; and the process of this patent is substantially superior to other potential carcinogen screening assays utilizing yeast or Salmonella.
Cancer is a disease affecting mammals; and an assay utilizing mammals is commonly believed to be superior to assays which utilize non-mammalian organisms (such as yeast, or Salmonella). Data generated from mammalian assays are generally more accepted than data generated from non-mammalian assays. One reason for this is that mammalian cells and body fluids contain enzymes some of which metabolize xeno-biotic agents and sometimes produce carcinogenic metabolites. These enzymes are not present to the same extent and in the same variety in non-mammalian systems; and, thus, to simulate a mammalian system with a non-mammalian assay mammalian liver homogenate must be added during the test. This not only adds a substantial amount of cost and time to the non-mammalian assay, but it is not clear that the mammalian liver homogenate accurately reflects the environment within the mammal.
There are several prior art processes for detecting the presence of carcinogens or potential carcinogens which utilize mammals; these are often referred to as "long term bioassays". In one such process a colony of mice is exposed to an agent and, after a substantial period of time, the occurrence of tumors is noted and evaluated. An example of such a process is disclosed in U.S. Pat. No. 4,736,866 of Leder, the disclosure of which is hereby incorporated into this specification. One of the problems with this type of process is the relatively long period of time which must be allowed for the tumor to develop after the mice have been exposed to the agent; after this period of time, each mouse in the test must be killed and subjected to an autopsy to evaluate the presence of the tumors. Furthermore, in this process, a large number of mice must be used. For instance, long term carcinogenesis studies require groups of 50 animals per dose for each of three doses, per sex for both sexes and per species for mice and rats. LD.sub.50 studies may require as many as 50 animals per dose, for four or more doses (see generally Lu Basic Toxicology, 2.sup.nd Edition, eds Taylor and Francis, Bristo, Pa., 1991). Additionally many of these assays require two years of exposure and one year or more of data gathering and evaluation for the long term carcinogenicity studies. Because of these factors, the assays are prohibitively expensive allowing only a few chemicals to be tested per year.
Further, for the long term studies high doses are required to obtain significant results (maximum tolerated dose experiments). This has led to widespread criticism of long term studies which culminated in questions about the validity of the entire program (e.g. Ames and Gold 1990; Weinstein 1991, "Carcinogenesis mechanism, the debate continues" letters, Science 252:902-904, 1991 and many public meetings to which both experts were invited, newspaper articles etc.; Abelson, 1994).
In an editorial, Abelson (1994) criticized the high dose experiments. The letters in response to this editorial (Science 266:1141-1145, 1994) are a good example of the current knowledge or lack thereof in the field. The only data that were cited in this debate are a limited number of examples on dose responses for carcinogenesis. A number of possible theories supporting or contradicting linear dose responses were cited without a single mechanistic example of data supporting those theories. This lack of data is currently splitting researchers into schools of different theories greatly limiting the public acceptance of current methods in risk assessments.
Furthermore, because of the high doses and the long term exposure, there is considerable public concern about the suffering of a large number of animals. In addition, in most cases a comparison of in vivo to in vitro results is not possible since different endpoints and different systems are being used. For many of these assays the measured endpoint is death, tumors per mouse etc. without much information about the mechanism of the toxicity. However, mechanistic information is required for our understanding of the toxic response which is essential for risk assessment and for prevention.
There is an assay which attempts to respond to the above criticisms, the "mouse spot test," which does not require the autopsies of a large number of-dead mice. This test is described in a review (Styles and Penman, 1985). In this test, a multiplicity of pregnant mice are exposed to the agent to be tested, and the offspring of such mice are then evaluated to determine the presence of spots in their coats. There is some correlation between the presence of such spots and the carcinogenicity of the agent tested. However the correlation is relatively poor.
As is indicated in U.S. Pat. Nos. 4,701,406 and 4,997,757, incorporated herein by reference, the well-known Ames assay (which utilizes certain mutant strains of bacteria) has several major disadvantages. Many classes of carcinogenic compounds consistently show poor responses in the Ames assay. The Ames assay is not very useful for evaluating certain metals, steroid hormones, and chlorinated hydrocarbons which although they are known to be carcinogens, give very poor responses in this assay. Also, the Ames assay is not generally useful for evaluating carcinogenic compounds which are not mutagenic; see, e.g., column 2 of U.S. Pat. No. 4,997,757.
It appears, however, that, notwithstanding the well-known shortcomings of the Ames assay, the mouse spot test is inferior to such assay. In several experiments (Styles and Penman, 1985) 45 known carcinogens and 6 known noncarcinogens were evaluated in both the Ames assay and the mouse spot test. The Ames assay correctly identified 84 percent of these agents; however, the mouse spot test correctly identified only 74 percent of these agents.
Currently, there is available a second mouse assay system: the "Big Blue.TM. Transgenic Mouse Mutagenesis Assay System," which is marketed by the Stratagene Company 11099 North Torrey Pines Road, La Jolla, Calif. According to Stratagene, this system utilizes a transgenic mouse lineage of the inbred strain C57BL/6 such that each cell of every mouse in the line contains multiple copies of a bacteriophage lambda shuttle vector which is approximately 43 kilobases in size (Mirsalis et al., 1993b).
The Stratagene system, however, has several distinct shortcomings. In the first place, it does not detect certain powerful carcinogens detectable by other assays. As shown by Mirsalis et al (1993b) transgenic B6C3F1 and C57BL/6 mice containing a lambda shuttle vector containing a lacI target do not detect the carcinogenic activity of methylmethane sulfate. (Methylmethane sulfate is a known hepatocarcinogen which does not induce mutation types which are detected by the Stratagene assay in the livers of such mice). Further, Mirsalis et al. (1993a) indicates that five daily administrations of carbon tetrachloride to such mice produced no increase in hepatic mutant frequency in Stratagene mice (lacI mouse).
The consensus among those in the molecular toxicology field appears to be that non-genotoxic carcinogens are not detectable by the Stratagene assay system. In Gunz et al. (at page 209) it is noted that "The negative results, both for lacI mutations in liver DNA and for the rate of hepatocyte division, show that the non-genotoxic carcinogens investigated do not give rise to a generally increased level of mutations or a sustained general increase in the rate of cell division".
It is known that the Stratagene mouse assay system is not very sensitive to ionizing radiation (Tao, et al. 1993). This is a critical shortcoming, since exposure to ionizing radiation constitutes a major health hazard. Thus, as was stated by J. Thacker (1992), "It is a sobering thought that, more than 60 years after the demonstration by Muller of the mutagenic effect of ionizing radiation, questions concerning the estimation of risk to the human population are still before us. This lack of progress is not through lack of effort but rather through the complexity of the task and the need to develop and refine methods of analysis".
Applicant has provided in U.S. Pat. No. 5,762,908 and assigned to the same assignee and incorporated herein in its entirety by reference, an improved method of evaluating carcinogens, including ionizing radiation, in mice. In the assay two alleles of a gene duplication, the pink-eyed unstable, (p.sup.un) mutation in the mouse are used to score for genomic rearrangement in response to exposure. The p.sup.un mutation causes a dilution of the pigment in coat-color and eye color and is due to a deletion disruption of the pink-eyed dilute locus creating a DNA sequence duplication of about 75 kb which is a head to tail duplication (Brilliant et al. 1991, Gondo et al. 1993). Reversion of this mutation is easily scorable as black spots on the dilute coat and is due to a deletion of one copy of the duplicated sequence resulting in production of wildtype melanin in melanocytes. The frequency of reversion of the p.sup.un mutation is uniquely sensitive towards the effects of x-rays. p.sup.un reversion events are also inducible by the Salmonella-assay-positive carcinogens EMS, MMS, ENU and benzo(a)pyrene as well as with the Salmonella-assay-negative carcinogens trichloroethylene, benzene and sodium arsenate. Most of these same chemicals are positive in the yeast deletion (DEL) assay (Schiestl 1989; Carls and Schiestl 1993) and those that have been tested are also positive in the human cell culture deletion assay.
However, while the p.sup.un reversion assay is a significant improvement as shown in the Examples of the co-pending application, '908 patent, it still has disadvantages. The assay is based on the induction of deletions in the embryo. Thus, the chemicals have to be able to enter through the placenta. Permeability of the placenta will differ from chemical to chemical. In addition, embryos may be much more sensitive to the toxic effects of chemicals and thus may die and be aborted rather than give scorable results. Further, quantitative PCR cannot be used to quantify deletion events which have been shown to be associated with the reversion phenotype in spontaneously revertant mouse (Gondo et al., 1993; Gardner et al., 1992). PCR also cannot be used for detection on the genomic level since p.sup.un contains a tandem duplication and the primers to detect the revertant would have to be placed on each side of the duplication in the unique sequence and it is not possible to amplify a 75 kb piece of DNA. Further, the p gene is only expressed in melanocytes, eyes, and gonades (Gardner 1992). Thus, tissue specific effects in other tissues cannot be studied.
It is an object of this invention to provide a toxicology assay utilizing mice which is capable of detecting the toxic effects of mutagenic carcinogens such as, e.g., methyl methane sulfonate, ethyl methane sulfonate, benzo(a)pyrene, ethylnitrosourea, and the like in any tissue in vivo in mice.
It is another object of this invention to provide a toxicology assay utilizing mice which is capable of detecting the toxic effects of nonmutagenic carcinogens such as, e.g., carbon tetrachloride, trichloroethylene, benzene, sodium arsenate, and the like in any tissue in vivo in adult mice.
It is an object of this invention to provide a mammalian assay for detecting potential carcinogens which utilizes mammals but does not require their autopsy.
It is yet another object of this invention to provide a mammalian assay for detecting potential carcinogens which can be completed in substantially shorter period of time than that required for long term bioassays.
It is yet another object of this invention to provide a mammalian assay for detecting potential carcinogens which, in at least some respects, is more accurate than prior art mammalian assays and more accurately mimics the human sensitivity.